Quinolinone or quinazolinone comprising antibiofilm compositions, compounds and methods and uses relating thereto

ABSTRACT

An antibiofilm composition comprising a compound of formula (A1), (A2) or (A3): 
                         
wherein each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  is independently selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, alkyl carboxy and amido; and X may be O or S, wherein the composition does not comprise a compound of formula (B1) or (B2):

This application is a divisional of U.S. patent application Ser. No.15/745,632, filed Jan. 17, 2018, which is a US National StageApplication under 37 CFR § 371 of PCT/EP2016/068355, filed Aug. 1, 2016,which application claims priority to UK Application No. 1513614.6, filedJul. 31, 2015. Each of these applications is incorporated herein byreference.

The present invention relates to methods of combating biofilms and inparticular biofilms caused by fungal infection. The invention alsorelates to compositions and novel compounds for use in such methods. Theinvention finds particular utility in combating fungal infections inmedical devices, for example implanted medical devices.

Implanted medical devices are extremely important and widely used inmodern medicine, saving lives and improving the quality of life ofmillions of people throughout the world. However negative outcomesfollowing implantation can occur due to microbial infection. Failure toeffectively treat such infections can lead to serious illness and evendeath. In many cases conventional therapies fail and the only treatmentoption is removal of the device.

Treatment of microbial infection with conventional antibiotics andantimycotics is increasingly difficult due to the continuing emergenceof resistant microbes.

Many pathogens can enter the biofilm mode of growth. A biofilm is formedwhen microbes form a structured community of cells. Biofilms play a keyrole in enabling a pathogen to overcome host defences and contribute toits virulence. Pathogens which enter the biofilm mode of growth mayemerge post treatment as resistant strains that can propagate freely andare recalcitrant to conventional therapies.

In addition to infection of biomedical implants, biofilm formationcontributes to other serious infections including antibiotic resistantinfections in hospitals and contamination of pharmaceuticalformulations.

The present invention seeks to provide means for disrupting biofilmformation and growth, and in particular biofilm formation due to fungalinfection. The present invention also provides means for combatingfungal infection, especially fungal infection due to Candida albicans orAspergillus fumigatus. Fungal infection can cause serious illness,especially in patients with compromised immune systems.

The development of antibiotics has slowed significantly in recent yearsand there is an increasing awareness of the hazards associated with theuse of antibiotic and chemical agents. There is thus an urgent need fornew, more effective therapies.

However when developing new therapeutic compounds it is important toconsider all effects that the compounds may have in a biologicalenvironment. For example some compounds are known to have antifungalproperties but are also known agonists for some bacterial species, forexample Pseudomonas aeruginosa. It is also necessary to ensure thatcompounds intended for medical use are not cytotoxic to human cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphically the XTT results for compounds B1, B2, methanol(i.e. the solvent alone) and untreated biofilms.

FIG. 2 shows the microscopy results for compounds C1, C2, C3, C4, C5,C6, C8, C10, C11 and C12.

FIG. 3 shows the microscopy results for compounds B1, B2, methanol anduntreated biofilms.

FIG. 4 shows the microscopy results for compounds C1, C2, C7, C8, C9 andC10.

FIG. 5A and FIG. 5B demonstrate how the compounds interrupt the biofilmrather than simply inhibiting growth.

According to a first aspect of the present invention there is providedan antibiofilm composition comprising a compound of formula (A1), (A2)or (A3):

wherein each of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is independently selectedfrom hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino,nitro, sulfoxy, thiol, carboxy, alkyl carboxy and amido; and X may be Oor S, wherein the composition does not comprise a compound of formula(B1) or (B2):

By carboxy substituent we mean to refer to the group COO⁻Y⁺ wherein Y isH⁺ or a metal or ammonium ion, for example Na⁺, K⁺ or NH₄ ⁺.

By alkyl carboxy substituent we mean to refer to the group COOR⁸ whereinR⁸ is alkyl. Preferred alkyl carboxy groups includes CO₂Me and CO₂Et.

By amido substituent we mean to refer to the group NHCOR⁹ where R⁹ is analkyl, alkenyl or aryl group. Preferably R⁹ is an alkyl group, suitablya C₁ to C₄ alkyl group.

When R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is an alkyl, alkenyl or arylsubstituent these groups may be straight chain or branched or maythemselves be substituted. Unsubstituted alky, alkenyll and aryl groupsare preferred.

Suitable alkyl, alkenyl and aryl groups may have up to 30 carbon atoms,preferably up to 20 carbon atoms, preferably up to 16 carbon atoms, forexample up to 12 carbon atoms or up to 8 carbon atoms.

When R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is an alkyl or alkenyl substituent itmay be straight chain or branched.

In some embodiments two substituents may be linked to form a cyclicalkyl or aryl group.

Each of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ may independently be halo.Suitable halo groups include fluoro, bromo and chloro. Chlorosubstituents are especially preferred.

Each of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ may be an alkoxy group. Preferredalkoxy groups are methoxy groups.

Suitably each of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is selected fromhydrogen, alkyl, alkoxy, halo and amino. Suitably each of R¹, R², R³,R⁴, R⁵, R⁶ and R⁷ is selected from hydrogen, C₁ to C₈ alkyl, methoxy,chloro and amino.

Preferably at least one of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ is nothydrogen.

R¹ may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy.

Preferably R¹ is selected from hydrogen, alkyl, alkoxy and halo.

Suitably R¹ is selected from hydrogen, methyl, methoxy and chloro.Preferably R¹ is hydrogen or methyl. Most preferably R¹ is hydrogen.

R² may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy.

Preferably R² is selected from hydrogen, alkyl, alkoxy, halo and alkylcarboxy.

Suitably R² is selected from hydrogen, C₁ to C₈ alkyl, methoxy, fluoro,bromo, chloro and CO₂R⁸ wherein R⁸ is C₁ to C₄ alkyl.

Suitably R² is selected from hydrogen, methyl, t-butyl, n-hexyl,methoxy, fluoro, bromo, chloro and CO₂Et. Preferably R² is selected fromhydrogen, methyl, n-hexyl, chloro and methoxy.

R³ may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy.

Preferably R³ is selected from hydrogen, alkyl, aryl and alkoxy.

Suitably R³ is selected from hydrogen, methyl and methoxy.

In some embodiments R³ and R⁴ may together form a group of formula—CH—CH—CH—CH— i.e., they form a further fused benzene ring. This furtherbenzene ring may itself be substituted.

Preferably R³ is hydrogen or methoxy.

R⁴ may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy.

Preferably R⁴ is selected from hydrogen, alkyl, aryl, halo and alkoxy.

Suitably R⁴ is selected from hydrogen, methoxy and chloro.

In some embodiments R³ and R⁴ together form a group of formula—CH—CH—CH—CH— i.e., they form a further fused benzene ring. This furtherbenzene ring may itself be substituted.

Preferably R⁴ is hydrogen or chloro.

R⁵ may be selected from hydrogen, alkyl, alkenyl aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy. Preferably R⁵ is hydrogen or alkyl.

When the invention comprises a compound of formula (A1) R⁵ may suitablybe hydrogen or alkyl. Preferred alkyl groups are C₁ to C₁₂ alkyl groups,preferably C₄ to C₁₂ alkyl, more preferably C₆ to C₁₀, for example C₇ toC₉ alkyl groups. These groups may be straight chain or branched and maybe substituted. Preferred are unsubstituted alkyl groups.

Preferably R⁵ is hydrogen or C₇ to C₉ alkyl. Suitably R⁵ is hydrogen.

When the invention comprises a compound of formula (A3) R⁵ may suitablybe hydrogen or alkyl. Preferably it is alkyl. Preferably R⁵ is C₁ to C₂₀alkyl, more preferably C₄ to C₁₂ alkyl, more preferably C₆ to C₁₀ alkyl,for example C₇ to C₉ alkyl. These alkyl groups may be straight chain orbranched and may be substituted. Preferred are unsubstituted alkylgroups.

R⁶ may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy and alkyl carboxy.

Preferably R⁶ is alkyl. Most preferably R⁶ is a C₁ to C₂₄ alkyl group,preferably a C₂ to C₁₆ alkyl group, more preferably a C₁₄ to C₁₆ alkylgroup, more preferably a C₄ to C₁₂ alkyl group, especially a C₆ to C₁₀alkyl group, for example a C₇ to C₉ alkyl group. The alkyl may bestraight chain or branched. It may itself be substituted. Preferably R⁶is an unsubstituted alkyl group. Preferably it is straight chain.Suitably R⁶ is an alkyl group of formula (CH₂)_(n)CH₃ wherein n is from3 to 15, preferably from 4 to 12, more preferably from 5 to 10, mostpreferably from 6 to 8. Preferably n is 6 or 8.

R⁷ may be selected from hydrogen, alkyl, alkenyl, aryl, halo, alkoxy,hydroxyl, amino, nitro, sulfoxy, thiol, carboxy, amido and alkylcarboxy.

When the invention comprises a compound of formula (A1) or (A3) R⁷ ispreferably hydrogen.

When the invention comprises a compound of formula (A2) R⁷ is preferablyhydrogen or amino (NH₂).

X may be O or S. Preferably X is O.

In some embodiments R¹ is hydrogen or methyl; R² is hydrogen, C₁ to C₈alkyl, halo or alkoxy; R³ is hydrogen, alkyl or alkoxy; R⁴ is hydrogenor halo; R⁵ is hydrogen or alkyl; R⁶ is alkyl; R⁷ is hydrogen or aminoand X is O.

In some embodiments R¹ is hydrogen; R² is hydrogen, methoxy or chloro;R³ is hydrogen or methoxy; R⁴ is hydrogen or chloro; R⁵ is hydrogen orC₄ to C₁₀ alkyl; R⁶ is C₅ to C₁₂ alkyl, preferably C₇ to C₉ alkyl; R⁷ ishydrogen or amino; and X is O.

In some preferred embodiments the composition comprises a compoundselected from:

(i) a compound of formula (A1) in which R¹ is hydrogen or methyl; R² ishydrogen, C₁ to C₆ alkyl, alkoxy, halo or CO₂Et; R³ is hydrogen, methylor methoxy or an aryl group with R⁴; R⁴ is hydrogen, methoxy, chloro oran aryl group with R³; R⁵ is hydrogen; R⁶ is C₆ to C₁₀ alkyl; and R⁷ ishydrogen;

(ii) a compound of formula (A2) wherein R¹ is hydrogen or methyl; R² ishydrogen, C₁ to C₆ alkyl halo or CO₂Et; R³ is hydrogen, methyl ormethoxy or an aryl group with R⁴; R⁴ is hydrogen, methoxy, chloro or anaryl group with R³; R⁶ is C₆ to C₁₀ alkyl and R⁷ is hydrogen or amino;and

(iii) a compound of formula (A3) wherein R¹ is hydrogen or methyl; R² ishydrogen, C₁ to C₆ alkyl, halo or CO₂Et; R³ is hydrogen, methyl ormethoxy or an aryl group with R⁴; R⁴ is hydrogen, methoxy, chloro or anaryl group with R³; R⁵ is C₆ to C₁₀ alkyl; R⁷ is hydrogen and X is O orS.

In some preferred embodiments the composition comprises a compoundselected from:

(i) a compound of formula (A1) in which R¹ is hydrogen or methyl; R² ishydrogen, C₁ to C₆ alkyl, chloro or methoxy; R³ is hydrogen; R⁴ ishydrogen or chloro; R⁵ is hydrogen; R⁶ is C₇ to C₉ alkyl; and R⁷ ishydrogen;

(ii) a compound of formula (A2) wherein R¹ is hydrogen; R² is hydrogen;R³ is hydrogen; R⁴ is hydrogen; R⁶ is C₇ to C₉ alkyl; and R⁷ is hydrogenor amino; and

(iii) a compound of formula (A3) wherein R¹ is hydrogen; R² is hydrogen;R⁴ is hydrogen; R⁵ is C₇ to C₉ alkyl; R⁷ is hydrogen and X is O.

Some especially preferred compounds for use in the invention structureshave the following structures:

Compounds (C1), (C2), (C4), (C5), (C6), (C7), (C8), (C9), (C10), (C11),(C12), (C13), (C14) and (C15) have been found to be advantageous due totheir reduced cytotoxicity towards mammalian cells.

Compounds (C1), (C2), (C3), (C4), (C5), (C6), (C7), (C8), (C9), (C10),(C11), (C13) and (C15) are particularly advantageous as, unlike someother compounds such as HHQ and PQS (i.e. the compounds of formula (B1)or (B2)), they do not increase the virulence of Pseudomonus aeruginosa.

Some preferred compounds for use in the present invention are thecompounds of formula (C1), (C2), (C5), (C13) and (C15).

Most preferred for use in the present invention are the compounds offormula (C1), (C2) and (C5).

The composition of the first aspect is an anti-biofilm composition. Byanti-biofilm composition we mean to include compositions that disrupt orremove biofilms or compositions that inhibit or prevent the growth ofbiofilm. Preferably the composition is effective against biofilms formedby fungi. Thus the present invention suitably provides compositions thatdisrupt or remove fungal biofilms or inhibit or prevent the growth offungal biofilms.

The anti-biofilm composition of the present invention may be used as asurface treatment composition. For example it may be contacted withsurfaces on which fungal growth is highly undesirable, for example in ahospital, especially a surgical environment.

By “effective against” we mean has anti-biofilm activity against, asdescribed above. The composition of the present invention is preferablyeffective against biofilms formed by fungal species selected from thegenera Candida and Aspergillus. Preferably the composition of the firstaspect is effective against biofilms formed by Candida albicans and/orAspergillus fumigatus.

In some embodiments the compositions of the present invention areeffective against Candida albicans. Some preferred compounds which areeffective against fungal biofilms formed by Candida albicans arecompounds of formula (A1), (A2) or (A3) wherein R¹ is hydrogen; R² ishydrogen, C₁ to C₆, alkyl or halo; R³ is hydrogen; R⁴ is hydrogen; R⁵ ishydrogen or C₆ to C₁₀ alkyl; R⁶ is C₆ to C₁₀ alkyl; R⁷ is hydrogen oramino and X is O.

Some especially preferred compounds for use against Candida albicansinclude

(i) a compound of formula (A1) in which R¹ is hydrogen; R² is hydrogen,methyl or chloro; R³ is hydrogen; R⁴ is hydrogen or chloro; R⁵ ishydrogen; R⁶ is C₇ alkyl; and R⁷ is hydrogen;

(ii) a compound of formula (A2) wherein R¹ is hydrogen; R² is hydrogen;R³ is hydrogen; R⁴ is hydrogen; R⁶ is C₇ alkyl and R⁷ is hydrogen oramino; and

(iii) a compound of formula (A3) wherein R¹ is hydrogen; R² is hydrogen;R³ is hydrogen; R⁴ is hydrogen; R⁵ is C₇ alkyl; R⁷ is hydrogen and X isO.

Some especially preferred compounds which are effective against fungalbiofilms formed by Candida albicans are compounds having the structures(C1), (C2), (C3), (C4), (C5) and (C6).

In some embodiments the compositions of the present inventions areeffective against Aspergillus fumigatus. Some especially preferredcompounds for use against Aspergillus fumigatus include compounds offormula (A1), (A2) or (A3) wherein R¹ is hydrogen; R² is hydrogen,alkoxy or C₁ to C₈ alkyl; R³ is hydrogen or alkoxy; R⁴ is hydrogen; R⁵is hydrogen; R⁶ is C₆ to C₁₀ alkyl; R⁷ is hydrogen or amino and X is O.

Compounds of formula (A1) or (A2) are especially effective againstAspergillus fumigatus. In some especially preferred compounds forcombating biofilms found by Aspergillus fumigatus include:

(i) a compound of formula (A1) in which R¹ is hydrogen; R² is hydrogen,n-hexyl or methoxy; R³ is hydrogen or methoxy; R⁴ is hydrogen or chloro;R⁵ is hydrogen; R⁶ is C₇ to C₉ alkyl; and R⁷ hydrogen; and

(ii) a compound of formula (A2) wherein R¹ is hydrogen; R² is hydrogen;R³ is hydrogen; R⁴ is hydrogen; R⁶ is C₇ alkyl and R⁷ is hydrogen oramino.

Some especially preferred compounds which are effective against fungalbiofilms formed by Aspergillus fumigatus are compounds having thestructures (C1), (C2), (C4), (C7), (C8), (C9), (C10), (C13) and (C15).

In some embodiments the composition of the first aspect may be apharmaceutical preparation comprising a compound of formula (A1), (A2)or (A3). Preferred features of the compound are as described above.

The pharmaceutical preparation may be provided in any suitable form. Forexample in some embodiments the pharmaceutical preparation may beprovided in the form of a gel, cream or paste suitable for topicalapplication.

In some embodiments the pharmaceutical preparation may be in the form ofa tablet, syrup or powder suitable for oral delivery.

In some embodiments the pharmaceutical preparation may be provided as asolution suitable for intravenous infusion or injection.

In some embodiments it is provided in a form suitable for inhalation.The pharmaceutical preparation may be provided in a form suitable fordelivery as a suppository or for subcutaneous delivery. It may beprovided on a patch. Other suitable forms will be known to the personskilled in the art.

The pharmaceutical preparation may include a diluent or carrier and oneor more further excipients. Suitable diluents, carriers and furtherexcipients will depend on the form of a pharmaceutical preparation andthe intended delivery method. The selection of suitable diluents,carriers and excipients is within the competence of the person skilledin the art.

The compound (A1), (A2) or (A3) is may suitably be included in apharmaceutical preparation in an amount of from 0.1 to 100 μg/ml, forexample from 1 to 50 μg/ml. However the selection of appropriate dosagerate and suitable delivery method will be within the competence of theskilled person.

The composition of the first aspect may be provided in many differentforms depending on the intended use thereof.

In some embodiments the composition may be a pharmaceutical preparationas defined above.

In some embodiments the composition may be a disinfectant composition. Adisinfectant composition may suitably be provided as a gel, paste,aerosol or solution. It may be provided as a powder, tablet or granulesor concentrated composition to be diluted prior to use.

In some embodiments the composition of the first aspect may be a coatingcomposition. Suitable ingredients for inclusion in such a compositionwill be known to the person skilled in the art.

According to a second aspect of the present invention there is provideda method of combating a biofilm at a locus, the method comprisingcontacting the locus with a composition of the first aspect.

By combating biofilms we mean to include disrupting or removing biofilmsand inhibiting or preventing the growth of biofilms. In particular themethod of the second aspect is a method of combating fungal biofilms.

By fungal biofilms we mean to include any biofilm which includes one ormore species of fungus growing in the biofilm mode. The fungal biofilmmay contain only a single fungal species or it may also contain one ormore other microbes, for example one or more further species of fungiand/or bacteria.

Preferred features of the second aspect are as defined in relation tothe first aspect and features described in relation to the second aspectalso apply to first aspect.

The method of the second aspect may be a method of disinfecting orsterilising a surface. Suitably the method disrupts any fungal biofilmattached to or growing on the surface. It may remove existing biofilms,disrupt existing biofilms and/or the method may prevent or inhibit theformation or growth of fungal biofilms at the surface.

In some embodiments the method of the second aspect may provide a methodof treating a medical device, the method comprising contacting thedevice with a composition of the first aspect. The method may be usedwith any suitable medical device, including surgical instruments,diagnostic equipment and implanted devices.

The composition may be contacted with the device by any suitable means.For example the composition may be sprayed, wiped or painted onto thedevice or the device could be immersed in the composition. In someembodiments the composition may provide a polymeric coating on thedevice.

For example the present invention may provide a method of disinfecting aclinical scope, for example an endoscope, the method comprisingcontacting the scope with a composition of the first aspect.

In some embodiments the method of the second aspect may be a method oftreating contact lenses, the method comprising immersing the contactlenses in a composition of the first aspect.

Thus the composition of the first aspect may be a contact lens solution.

It is currently very difficult to eliminate fungal biofilm growth oncontact lenses using conventional antimicrobial agents. This can lead tocontact lens wearers developing very uncomfortable fungal eyeinfections. Thus the method of the present aspect of the invention ishighly beneficial.

The method of the second aspect is a method of combating biofilms,preferably fungal biofilms at a locus. The locus may be the surface ofan inanimate object—or it may be a location within the body of ananimal, suitably a human.

The method of the second aspect suitably involves combating fungiselected from Candida albicans and/or Aspergillus fumigatus.

The composition of the first aspect of the present invention may be usedin a wide variety of applications. The amount of the compounds offormula (A1), (A2) or (A3) that should be included in the compositionand the further components of the composition will depend on theintended use of the composition. However the selection of appropriateamounts of the active compounds and suitable further components iswithin the competence of the person skilled in the art.

According to a third aspect of the present invention there is provided acompound of formula (A1), (A2) or (A3) for use in the treatment of afungal biofilm infection. Preferred features of the third aspect are asdefined in relation to the first and second aspects.

Current treatment options for fungal biofilm-related infections arelimited by the intrinsic tolerance of biofilms to anti-fungalantibiotics, otherwise known as antimycotics, whereby the biofilmeffectively shields the microbe from the active agent. In addition,changes to the physiology of the cell within the biofilm can alsocontribute to the intrinsic resistance reported towards antibiotics.While there has been some limited success with conventional antibiotics,e.g. liposomal amphotericin B, reports of resistance continue to emergewith the biofilm mode of growth a primary factor underpinning resistanceto conventional antimicrobial agents.

The compositions of the present invention help to disrupt biofilms orprevent or inhibit the growth of biofilms. This may allow conventionalagents to attack microbes and kill them.

Thus the present invention may provide a combination of a compound offormula (A1), (A2) or (A3) and one or more further antimicrobial agentsfor use in the treatment of a fungal biofilm infection.

The composition of the first aspect may in some embodiments comprise acompound of formula (A1), (A2) or (A3) and one or more furtherantimicrobial agents.

Suitably the one or more further antimicrobial agents is an antifungalagent. Suitable antifungal agents include fluconazole, amphotericin Band nystatin.

The present invention may provide a compound of formula (A1), (A2) or(A3) for use in the treatment of a fungal biofilm infection caused by afungus selected from Aspergillus fumigatus, Candida albicans and/ormixtures thereof.

The third aspect of the present invention may provide a compound offormula (A1), (A2) or (A3) for use in the treatment of a fungal biofilminfection caused by Aspergillus fumigatus. Compounds particularlyeffective for use in the treatment of a fungal biofilm infection withAspergillus fumigatus are as defined in relation to the first aspect.

The third aspect of the present invention may provide the use of acompound of formula (A1), (A2) or (A3) for use in the treatment of afungal biofilm infection caused by Candida albicans. Compoundsespecially suitable for use in such treatment are as defined in relationto the first aspect.

As such the present invention provides a significant advancement in thepotential prevention of serious illness and possibly fatalities due toinfection with fungal biofilms. Fungal biofilms caused by Candidaalbicans and Aspergillus fumigatus can lead to serious infections inimplanted medical devices. They are also commonly associated withhospital acquired infections and contamination of pharmaceuticalformulations. For example biofilms on medical devices used to treat morethan one patient may not be effectively removed by conventionalsterilisation techniques.

Infection with fungi that form biofilms can cause very serious illnessand even death, especially in patients with other conditions and/orweakened immune systems. Aspergillus fumigatus for example can formbiofilms within the lungs of a patient. Once the biofilm has formed itis very difficult for the infection to be treated. Conventionalantimicrobial agents are often ineffective in such circumstances.

However the compositions of the present invention have been found to beable to disrupt fungal biofilms and thus may be used in the treatment ofpatients having a fungal biofilm infection, for example associated witha respiratory disease. Patients with, for example, cystic fibrosis arevulnerable to infection with fungal biofilms.

Thus the present invention may provide compounds of formula (A1), (A2)and (A3) for use in the treatment of a respiratory disease.

In some embodiments the invention may provide compounds of formula (A1),(A2) and (A3) for use in the treatment of cystic fibrosis.

The invention may suitably provide compounds of formula (A1), (A2) or(A3) for use in the disruption or prevention of a fungal biofilminfection.

The present invention may be useful in the treatment of infectionscaused by fungal biofilms. The invention may also be used to prevent orinhibit the growth of fungal biofilms. Thus the present invention mayprovide a prophylactic treatment.

According to a fourth aspect of the present invention there is provideda medical device having a coating on at least a portion thereof; whereinthe coating comprises a compound of formula (A1), (A2) or (A3).

Preferred features of the fourth aspect are as defined in relation tothe first, second and third aspects.

The medical device of the present invention may be selected from a widerange of medical devices. Suitable medical devices include prostheticlimbs, hip replacement joints, stents, catheters, medical scopes andcontact lenses.

According to a fifth aspect of the present invention there is provided acompound of formula (A3):

Preferred features of the fifth aspect are as defined in relation to thefirst, second, third and fourth aspects.

According to a sixth aspect of the present invention there is provided amethod of preparing a compound of formula (A3).

Any suitable method may be used. One suitable method involves stirringan anthranilic acid with an alkyl halide (for example bromoheptane) inbase at elevated temperature. The resultant secondary amine product isheated with acetic anhydride in acid. Work-up provides the crude2-quinolones which may be purified by recrystallization.

In an alternative method an aniline may be heated with dimethylsulphatein base and an alkylhalide (for example bromoheptane) was added. Theresultant secondary amine product may be heated with Meldrum's acidfollowed by addition of Eaton's reagent. Work-up and purification usingchromatography gives the target 2-quinolones of formula (A3).

According to a seventh aspect of the present invention there is provideda compound of formula (A3) for use in therapy.

The present invention may further provide compounds of formula (A3) foruse in the treatment of a fungal biofilm infection.

Suitably the present invention provides compounds of formula (A3) foruse in the treatment of a biofilm infection caused by Aspergillusfumigatus and/or Candida albicans.

In some embodiments the present invention provides compounds of formula(A3) for use in the treatment of a biofilm infection caused by Candidaalbicans.

The present invention will now be further described with reference tothe following non-limiting examples.

EXAMPLE 1

The following compounds were prepared using the methods described inOrg. Biomol. Chem., 2015, 13, 5537-5541 and analogous processes:

The properties of these compounds were tested and compared to theproperties of:

The following general conditions were used in the biological testing ofsome of the above compounds to assess their effectiveness againstCandida albicans:

C. albicans Stock Maintenance and Culturing Conditions.

C. albicans strain SC5314 was sub-cultured from 15% (v/v) glycerolstocks at −80° C. onto Yeast Peptone Dextrose (YPD) medium [1% (w/v)yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose] and incubated at30° C. overnight.

P. aeruginosa Stock Maintenance and Culturing Conditions.

P. aeruginosa strains, PAO1 and pqsA mutant, containing thechromosomally inserted pqsA-lacZ promoter fusion on plasmidpUC18-mini-Tn7, were sub-cultured from glycerol stocks onto LB agarplates, supplemented with Carbenicillin (200 μg/ml) and X-gal (40μg/ml), and incubated at 37° C. overnight. Single colonies wereinoculated into LB broth (20 ml), supplemented with Carbenicillin (200μg/ml), and incubated at 37° C., shaking at 180 rpm overnight. Forsubsequent experiments, the OD_(600 nm) was recorded and a startingOD_(600 nm) of 0.02 was inoculated into fresh LB broth, supplementedwith Carbenicillin (200 μg/ml) and incubated at 37° C., shaking at 180rpm.

Test Compounds

The test compounds in desiccated form were re-suspended in Methanol tocreate a 10 mM stock. A working concentration of 100 μM was used in allexperiments.

TLC Analysis.

Silica TLC plates, activated by soaking in 5% (w/v) K₂HPO₄ for 30 minwere placed in an oven at 100° C. for 1 hr. Test compounds (5 μl, 10 mM)were spotted approximately 1 cm from the bottom. The spots were driedand the plate placed in a mobile phase comprising 95:5dichloromethane:methanol. The plate was viewed under UV light when themobile phase had run 5 cm below the top of the plate.

EXAMPLE 2

The ability of some of the compounds detailed in example 1 to disruptCandida albicans was tested using Confocal Scanning Laser Microscopy andan XTT Metabolic Assay. The XTT assay is a commonly used quantitativemethod assessing Candida biofilm mass and growth often in response tonovel drug therapies.

C. albicans biofilm formation was carried out in 96 well plates by amethod of the prior art. Briefly, C. albicans was inoculated into YeastNitrogen Base [10% (w/v)] and glucose/maltose [10% (w/v)] and incubatedovernight at 30° C. Seeding densities for all subsequent experiments(N=3) were OD₆₀₀=0.05. Cells were grown in Yeast Nitrogen Basesupplemented with 1 M Phosphate buffer and acetyl-D-glucosamine (YNB-NP)along with appropriate volumes of test compound to provide the desiredconcentration. Cells were cultured in 96 well plates for 1 hr at 37° C.to facilitate yeast attachment. After this incubation, the supernatantswere aspirated, the wells washed twice with YNB-NP media, and freshmedia with the same concentration of test compound added to theappropriate wells. The plate was incubated for 24 hr at 37° C. The nextday, the cultures were aspirated and the wells washed once with YNB-NPmedia.

C. albicans biofilm quantification was carried out in 96 well platesusing a semi-quantitative Tetrazolium salt,2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carboxanilideinner salt (XTT) reduction assay (Hawser, Tunney et al, AntimicrobAgents Chemother. 2004 May; 48(5):1879-81). XTT (0.01 g) was dissolvedin sterile water (20 ml) and filter sterilized. 10 μl of Menadionedissolved in Acetone was added to the XTT solution, just before use. TheXTT-Menadione Solution (100 μl) was added to each well. The plate wasincubated in the dark at 37° C. for up to 2 hr to allow for colourdevelopment. The OD_(492 nm) was recorded for each well. Experimentswere repeated at least three times, with at least eight technicalreplicates.

For the microscopy experiments C. albicans biofilms were grown on glasscoverslips in 6 well plates, using the biofilm formation protocol above.Briefly, an overnight culture of C. albicans was added to YNB-NP mediato give an OD₆₀₀=0.05. Test compounds were added at appropriateconcentrations. The 6 well plates were incubated at 37° C. for 1 hr tofacilitate attachment after which they were washed once in YNB-NP andthen fresh test compound in YNB-NP added. Plates were incubatedovernight at 37° C. Next day, glass coverslips were washed once inYNB-NP and stained. Calcofluor (1 mg/ml) and 10% (w/v) potassiumhydroxide were added drop-wise to coverslips, washed in PBS and viewed.Concavalin A and FUN-1 were added at 50 μg/ml in 1 ml PBS and incubatedat 37° C. for 30 mins. Coverslips were washed in PBS and viewed. Allimaging was carried out on a Zeiss LSMS confocal microscope. Confocalimages were recorded under a bright field lens using ×20 objectivemagnification. Filter cubes facilitating fluorescent imaging were usedto record images for Calcofluor at 405 nm, Con A at 488 nm and FUN-1 at543 nm. All images were captured using the Zeiss HBO-100 microscopeilluminating system, processed using the Zen AIM application imagingprogram and converted to JPGs using Axiovision 40 Ver. 4.6.3.0. Aminimum of three independent biological repetitions were carried out.

Experiments were first carried out using the test compound atconcentration of 100 μM.

FIG. 1 shows graphically the XTT results for compounds B1, B2, methanol(i.e. the solvent alone) and untreated biofilms.

FIG. 2 shows the microscopy results for compounds C1, C2, C3, C4, C5,C6, C8, C10, C11 and C12. The results of the XTT tests for thesecompounds are detailed in table 1:

TABLE 1 Absorbance at 492 nm Compound Mean Standard deviation Untreated0.851 0.079 MeOH (control) 0.647 0.007 B1 0.200 0.027 B2 0.999 0.120 C10.162 0.021 C2 0.178 0.003 C3 0.307 0.062 C4 0.302 0.054 C5 0.242 0.029C6 0.336 0.067 C8 0.457 0.014 C10 0.419 0.001 C11 0.242 0.041 C12 0.2980.029

The above experiments were repeated, this time using concentrations oftest compound of 100 μM, 50 μM and 10 μM to show that the compounds canstill be effective at lower concentrations. The results are shown intable 2.

TABLE 2 Compound Mean Standard Deviation MeOH (control) 1 0 B1 0.450.047 B2 1.2 0.005 C1 10 μM 0.93 0.011 C2 10 μM 0.68 0.081 C3 10 μM 0.710.024 C4 10 μM 0.86 0.018 C10 10 μM 0.58 0.006 C1 50 μM 0.72 0.011 C2 50μM 0.38 0.061 C3 50 μM 0.57 0.001 C4 50 μM 0.67 0.051 C10 50 μM 0.500.010 C1 100 μM  0.58 0.001 C2 100 μM  0.25 0.055 C3 100 μM  0.51 0.010C4 100 μM  0.58 0.040 C10 100 μM  0.53 0.114

EXAMPLE 3

The cytotoxicity of compounds B1, B2, C2, C3, C4, C5, C6 and C12 wastested according to the following method:

Lactate dehydrogenase (LDH) release from IB3 lung epithelial cells wasassayed as a measure of cytotoxicity using an LDH colorimetric kit(Roche) according to manufacturers' instructions. Briefly, IB3-1 cellswere seeded onto 96 well plates and treated with methanol (control) andtest compounds (100 μM). Following 16 hr incubation at 37° C. and 5%CO₂, supernatants were removed and added to catalyst reaction mixture ina fresh plate and further incubated at 37° C. and 5% CO₂ for 30 mins toallow for colour development. After this period, the plate was analysedon an ELISA plate reader at OD_(490 nm). Cytotoxicity was expressed as apercentage of cells treated with 0.1% (v/v) Triton (100% cytotoxicity).The results are detailed in table 3:

TABLE 3 % Cytotoxicity Compound Mean Standard deviation MeOH 3.844 1.40B1 38.93 10.89 B2 13.84 4.18 C2 28.94 6.76 C3 23.77 3.46 C4 15.46 4.35C5 31.04 7.74 C6 19.70 10.20 C12 16.68 3.97

The above experiments were repeated, testing some compounds againstfurther cell lines (A549, DU145 and HeLa). The results are detailed intable 4:

TABLE 4 Cytotoxicity Standard Cell Line Compound Mean Deviation A549 C18.739 1.046 A549 C12 32.37 6.63 DU145 C1 6.26 4.52 DU145 C3 32.42 14.27DU145 C4 24.42 6.49 DU145 C12 8.28 1.43 DU145 C10 31.7 5.43 HeLa C115.96 11.59 HeLa C12 34.57 19.08

EXAMPLE 4

The virulence of compounds B1, B2, C2, C3, C4, C5, C6, C8 and C11towards P. aeruginosa was assessed using the following method:

RNA Isolation and qRT-PCR Transcriptional Analysis.

Overnight Candida albicans cultures were diluted to 0.05 at OD₆₀₀ ineither YNB or YNB-NP (Difco). YNB cultures were supplemented withmethanol whereas YNB-NP cultures were supplemented with either 10 mM HHQor the methanol volume equivalent. Cultures were grown at 30° C. withagitation (180 rpm) for 6 hours after which they were centrifuged at4000 rpm, supernatants discarded and pellets frozen at −20° C. untilprocessing. RNA was isolated using the MasterPure Yeast RNA purificationkit (Cambio Ltd, Cambridge UK) according to manufacturersspecifications, and was quantified using a ND-1000 Spectrophotometer(NanoDrop Technologies, USA). Genomic DNA was enzymatically removedusing Turbo DNA-free DNase (Ambion), and samples were confirmed DNA freeby PCR. RNA was converted to cDNA using random primers and AMV reversetranscriptase (Promega) according to manufacturers instructions. qRT-PCRwas carried out using the Universal ProbeLibrary (UPL) system (Roche)according to manufacturers specifications, and samples were normalisedto C. albicans actin transcript expression (ACT1).

Phenazine Extraction.

P. aeruginosa strains were cultured as described above for 24 hr, withthe addition of test compounds (10 μM). Cultures were centrifuged at4000 rpm for 10 minutes and the cell free supernatant (5 ml) removed.Chloroform (3 ml) was added, and mixed by vortex. After centrifugationat 4000 rpm for 5 mins, the lower aqueous phase was transferred to 0.2 MHCl (2 ml). Samples were mixed by vortex and centrifuged at 4000 rpm for5 mins to separate the phases. An aliquot of the top phase (1 ml) wasremoved and spectrophotmetrically analysed at OD_(570 nm). Phenazineproduction was calculated using the following formula:OD_(570 nm)×2×17.072 and the units expressed in μg/ml.

Promoter Fusion Based Expression Analysis.

Promoter fusion analyses were performed in a 96-well format. Briefly,overnight cultures of wild-type PAO1 pqsA-lacZ (pLP0996) and mutantstrain PAO1 pqsA⁻ pqsA-LacZ were diluted to OD_(600 nm)=0.02 in LB. Testcompounds at 100 μM final concentration were added, mixed, aliquotedinto 96 well plates and incubated overnight at 37° C. with shaking. Thenext day, OD_(600 nm) values were recorded in a plate reader. Aliquotsof cells (0.02 ml) were permeabilised [100 mM dibasic sodium phosphate(Na₂HPO₄), 20 mM KCl, 2 mM MgSO₄, 0.8 mg/mL CTAB(hexadecyltrimethylammonium bromide), 0.4 mg/mL sodium deoxycholate, 5.4μL/mL beta-mercaptoethanol] and added to substrate solution [60 mMNa₂HPO₄, 40 mM NaH₂PO₄, 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG),2.7 μL/mL β-mercaptoethanol]. The kinetics of colour development wasmonitored and the reactions were stopped using 1M NaCO₃. OD_(420 nm)were recorded as above. Miller units were calculated using the followingequation; 1000×[OD_(420 nm)/(OD_(600 nm))×0.02 ml×reaction time (mins)].

The phenazine production levels and Miller units in the PAO1pqsA-pqsA-lacZ strain are detailed in table 5.

TABLE 5 Phenazine (μg/ml) Miller Units Standard Standard Compound Meandeviation Mean deviation Untreated 0.224 0.525 11711 4153 Methanolcontrol 0.080 0.131 12287 4283 Water control 3.088 0.799 — — B1 2.6970.537 65076 27746 B2 2.117 0.727 61429 28389 C2 0.541 0.497 16027 4749C3 0.774 0.523 18350 4331 C4 0 0 35489 12432 C5 0 0 11940 691 C6 0.0110.019 16678 9700 C8 0.205 0.354 11753 1695 C11 0.432 0.603 12254 1913

The virulence of compounds B2, C13 and C15 towards P. aeruginosa wasassessed using an analogous method. The results are shown in table 6:

TABLE 6 Normalised Phenazine (μg/ml) Normalised Miller Units StandardStandard Compound Mean Deviation Mean Deviation Methanol control 1 1DMSO control 1.32174 0.522353 1.44997 1.08499 B2 4.196486 1.8027233.078914 0.850754 C13 0.747026 0.160539 0.371541 0.173011 C15 0.4983710.319781 0.467063 0.154079

Data (a minimum of n=2 for each datapoint) is presented normalized tothe methanol control.

EXAMPLE 5

The ability of some of the compounds detailed in example 1 to disruptAspergillus fumigatus was tested.

Aspergillus fumigatus Stock Maintenance and Culturing Conditions.

A. fumigatus Af293 was routinely grown on Sabouraud dextrose agar (SDA)in 100 ml cell culture flasks at 37° C. for 3-4 days until a lawn offungal growth was observed.

Spore Capture and Biofilm Assay

A. fumigatus Af293 was grown on SDA in 100 ml cell culture flasks at 37°C. for 3-4 days. Conidia were harvested by flooding the surface of theagar plates with 5 ml PBS (Oxoid) containing 0.025% (v/v) Tween 20 andgently moving the liquid over the surface of the fungal lawn. Theconidial suspension was transferred into a 25 ml sterile container andconidia were counted using a Neubauer haemocytometer and lightmicroscope. Conidia were adjusted to the required concentration in RPMI1640 (Sigma) buffered to pH 7.0 with 0.165 M MOPS immediately prior tobiofilm formation analysis.

To assess biofilm formation, counted A. fumigatus spores (1×10⁵) in MOPSbuffered RPMI 1640 were inoculated into 24- and 96-well plates and grownovernight in the presence of 100 μM HHQ, PQS and the test compounds.After 24 hrs, the media was removed and the biofilm washed twice withdistilled water after which 0.1% crystal violet (CV) was added to eachwell and allowed to stand for 1 hr at room temperature. The crystalviolet was removed and all wells washed in a water bath by inversion.Ethanol was added to each well to solubilise biofilms after whichsamples were read on a spectrophotometer at Abs_(595 nm).

Staining and Microscopy

A. fumigatus spores (1×10⁵) were inoculated onto glass coverslips in 24well plates and grown in the presence of analogues overnight at 37° C.in RPMI buffered with MOPS pH 7.0. After 24 hrs, glass coverslips werewashed once in PBS and stained. Staining and microscopy was performed asdescribed in Example 2.

FIG. 3 shows the microscopy results for compounds B1, B2, methanol anduntreated biofilms.

FIG. 4 shows the microscopy results for compounds C1, C2, C7, C8, C9 andC10. The results of the XTT tests are detailed in table 7:

TABLE 7 Relative absorbance at 595 nm compared to methanol controlCompound Mean Standard deviation Methanol 1 0 C1 0.39 0.18 C2 0.37 0.10C7 0.27 0.14 C8 0.47 0.16 C9 0.18 0.05 C10 0.24 0.12 C13 0.38 0.09 C140.42 0.21 C15 0.25 0.10

The above experiments were carried out using biofilms grown fromlaboratory sources of A. fumigatus. The experiments were repeated totest some compounds against biofilms grown from three different clinicalsources of A. fumigatus (CFBRC1, CFBRC2 and CFBRC3. The results areshown in table 8:

TABLE 8 Compound Mean Standard Deviation CFBRC1 C1 0.432 0.091 CFBRC1 C20.310 0.190 CFBRC1 C5 0.297 0.171 CFBRC1 C9 0.593 0.126 CFBRC1 C10 0.7720.267 CFBRC1 C13 0.507 0.181 CFBRC1 C14 0.550 0.068 CFBRC1 C15 0.5180.287 CFBRC1 B1 0.412 0.160 CFBRC1 B2 0.561 0.325 CFBRC1 MeOH 1 0 CFBRC2C1 0.497 0.147 CFBRC2 C2 0.304 0.147 CFBRC2 C5 0.359 0.170 CFBRC2 C90.484 0.040 CFBRC2 C10 0.567 0.157 CFBRC2 C13 0.620 0.088 CFBRC2 C140.823 0.246 CFBRC2 C15 0.443 0.280 CFBRC2 B1 0.509 0.204 CFBRC2 B2 0.6730.274 CFBRC2 MeOH 1 0 CFBRC3 C1 0.471 0.182 CFBRC3 C2 0.292 0.161 CFBRC3C5 0.483 0.247 CFBRC3 C9 0.439 0.129 CFBRC3 C10 0.670 0.018 CFBRC3 C130.609 0.108 CFBRC3 C14 0.501 0.073 CFBRC3 C15 0.413 0.217 CFBRC3 B10.484 0.187 CFBRC3 B2 0.546 0.338 CFBRC3 MeOH 1 0

EXAMPLE 6

The anti biofilm properties of some of the claimed compounds was testedby carrying out a viable biofilm assay and measuring the growth of themicrobes.

Viable Colony Biofilm Assay

C. albicans biofilms, supplemented with test and comparative compounds,were grown in 6-well plates and incubated overnight at 37° C. Briefly,C. albicans Yeast Nitrogen Base (YNB) cultures were measured at OD600nm, diluted to 0.05 in YNB-NP supplemented with analogues, plated onto6-well plates and incubated for 1 hr at 37° C. Media was removed, wellswere washed twice with sterile PBS and supplemented with fresh YNB-NPwith analogues. Plates were incubated overnight at 37° C. after whichmedia was removed and wells washed with sterile PBS. For serialdilutions, biofilms were cell-scraped into 1 ml PBS, vortexed, andserially diluted into sterile PBS. Serial 147 dilutions were plated (100μl) onto YPD agar and incubating overnight at 37° C. Colonies werecounted and recorded the next day.

C. albicans Growth Curves

Overnight C. albicans cultures grown in YNB were diluted to 0.05 in YNBsupplemented with analogues. Cultures (200 μl) were added to each wellof a 100 well plate and grown for a 24 hr period on a Bioscreen Cspectrophotometer (Growth Curves USA).

The results, shown in FIGS. 5A and 5B demonstrate how the compoundsinterrupt the biofilm rather than simply inhibiting growth.

The invention claimed is:
 1. A method of combating a biofilm at a locus,the method comprising contacting the locus with a composition comprisinga compound of formula:


2. A method according to claim 1 wherein combating a biofilm involvesdisrupting or removing a biofilm or inhibiting or preventing the growthof a biofilm.
 3. A method of combating a fungal biofilm at a locus, themethod comprising contacting the locus with a composition comprising acompound of formula (A1), (A2), or (A3):

wherein each of R¹, R², R³, R⁵, R⁴, R⁶, and R⁷ is independently selectedfrom hydrogen, alkyl, alkenyl, aryl, halo, alkoxy, hydroxyl, amino,nitro, sulfoxy, thiol, carboxy, alkyl carboxy, and amido.
 4. A methodaccording to claim 3 which is a method of combating a fungal biofilmcomprising a single fungal species or which contains one or more othermicrobes, for example one or more further species of fungi and/orbacteria.
 5. A method according to claim 1 which is a method ofdisinfecting or sterilizing a surface.
 6. A method according to claim 1which is a method of treating a medical device.
 7. A method according toclaim 1 which is a method of treating contact lenses, the methodcomprising immersing the contact lenses in a composition comprising acompound of formula C1.
 8. A method according to claim 3 which involvescombating fungi selected from Candida albicans and/or Aspergillusfumigatus.
 9. A method of combating a biofilm at a locus, the methodcomprising contacting the locus with a composition comprising a compoundof formula (A2):

wherein R¹ is hydrogen or methyl; R² is hydrogen, C₁ to C₈ alkyl, haloor alkoxy; R³ is hydrogen, alkyl or alkoxy; R⁴ is hydrogen or halo; R⁶is alkyl; and R⁷ is hydrogen or amino.
 10. A method according to claim 9wherein the composition contacted with the locus comprises a compound offormula (A2) wherein R¹ is hydrogen; R² is hydrogen; R³ is hydrogen; R⁴is hydrogen; R⁶ is C7 to C9 alkyl; and R⁷ is hydrogen or amino.
 11. Amethod according to claim 9 wherein the composition contacted with thelocus is effective against Candida albicans and comprises a compound offormula (A2) wherein R¹ is hydrogen; R² is hydrogen; R³ is hydrogen; R⁴is hydrogen; R⁶ is C7 alkyl and R⁷ is hydrogen or amino.
 12. A methodaccording to claim 9 wherein the composition contacted with the locus iseffective against Aspergillus fumigatus and comprises a compound offormula (A2) wherein R¹ is hydrogen; R² is hydrogen; R³ is hydrogen; R⁴is hydrogen; R⁶ is C7 alkyl; and R⁷ is hydrogen or amino.
 13. A methodaccording to claim 1 which is a method of treating a fungal biofilminfection.